The present invention, in some embodiments thereof, relates to isolated populations of renal stem cells and methods of isolating and using same.
The kidney is a vital organ in mammals, responsible for fluid homeostasis, waste excretion, and hormone production. There are a variety of possible injuries and disorders including cancer, trauma, infection, inflammation and iatrogenic injuries or conditions that can lead to chronic disease or cause reduction or loss of function of a kidney. The incidence of chronic kidney disease in the United States has reached epidemic proportions, and a significant number of these patients will develop end-stage renal disease (ESRD), with glomerular filtration rates too low to sustain life. Dialysis is the major treatment modality for ESRD, but it has significant limitations in terms of morbidity, mortality, and cost. Allogenic kidney transplantation provides significant benefits in terms of mortality and is ultimately less costly, but is hampered by a severe shortage of available donor organs. Acute renal failure (ARF) is also quite common, having a mortality rate that ranges from 20 to 70%. For a number of reasons, including aggressive care of an older patient population, the mortality rate due to ARF has not changed over the past 20 years despite advances in technology and therapies.
Although kidney disease has a variety of individual types, they appear to converge into a few pathways of disease progression. The functional unit of the kidney is the nephron. There is a decrease in functioning nephrons with the progression of the disease; the remaining nephrons come under more stress to compensate for the functional loss, thereby increasing the probability of more nephron loss and thus creating a vicious cycle. Furthermore, unlike tissues such as bone or glandular epithelia which retain significant capacity for regeneration, it has generally been believed that new nephron units are not produced after birth, that the ability of the highly differentiated tissues and structures of the kidneys have limited reparative powers and, therefore, that mammals possess a number of nephron units that can only decline during post-natal life. There is an increasing interest in developing novel therapies for kidney disease, including artificial organs, genetic engineering, and cell therapy.
The early development of the mammalian metanephros, the direct precursor tissue of the adult kidney, is a complex process that involves highly regulated interactions between two derivatives of the intermediate mesoderm, the wolffian duct and the metanephric/nephrogenic mesenchyme. Reciprocal signaling between the neohrogenic/metanephric mesenchyme and a derivative of the nephric duct known as the ureteric bud results in branching of the ureteric bud (UB) and condensation of metanephric mesenchyme (MM) at its tips (4, 5). The condensed mesenchyme is thought to form a precursor cell population, which both maintains itself at the tips of the UB (via proliferation and/or addition from the surrounding non-condensed mesenchyme) and gives off cells that differentiate into nephrons, the functional filtration unit of the kidney (6). Recent experiments have established that the progenitor cell in the MM fulfils the criteria of a true committed stem cell in that is capable of self-renewing and of differentiating towards different types of nephron epithelia (7-9).
The human metanephros appears at the 5th week of gestation and renal stem/progenitor cells in the nephrogenic mesenchyme are induced to form nephrons until 34 weeks of gestation (4, 6). For renal regeneration, both human precursor tissue (10-12) or fetal kidney cell transplantation (13, 14) can be utilized. Isolation of specific human renal progenitors from the nephrogenic mesenchyme requires the characterization of surface markers that would enable cell collection. Given the cellular heterogeneity in the developing human kidney (6), eliminating the unwanted mature cell populations from further cultivation steps, prior to transplantation, would increase the purity of the graft and allow for a better defined cell composition to be transferred.
While the transcriptional program specifying a renal progenitor cell has been thoroughly contemplated (15) corresponding cell surface markers have been hardly studied. Recently, the present inventors performed microarray studies of the human kidney, including adult (AK) and fetal kidneys (FK) and their corresponding tumors, renal cell carcinoma (RCC) and wilms' tumor (WT) (16). Wilms' tumor is classified as a primitive, multilineage malignancy of embryonic renal precursors that are arrested in different stages of differentiation, thus forming in the tumor a cell population similar to condensed mesenchyme (blastema) and also mature epithelial/tubular and stromal cells (17). While fetal kidneys were heterogeneous, WT xenografts were used that by serial passage in mice were highly enriched for blastema at the expense of differentiated elements (16, 18). Genes that were up-regulated in both the stem-like WT xenografts and the human FK were sought, as these were suggested to characterize the progenitor population arising from the MM (‘progenitor’ genes). Among these were the transcription factors specifying the kidney progenitor cells (7, 15, 19, 20) including WT1, PAX2, LIM1, SIX1, EYA1, SALL1, and CITED1. In addition, various cell surface markers were detected, including NCAM1, ACVRIIB, FZD2, FZD7, GPR39, NTRK2 and DLK1/PREF (16).
U.S. Patent Application 20020102241 discloses Flk-1 positive/Sca-1 negative adult renal stems cells and uses thereof. The cells are described as useful for the regeneration of damaged kidney tissue, the generation of artificial kidneys and the delivery of transgenes.
U.S. Patent Application 20050260623 discloses the identification of adult human stem cells including adult renal stem cells by detecting the expression of Oct-4, and the lack of GJIC activity.
U.S. Patent Application 20070065942 provides human renal stem cells. Also described are human renal stem cells isolated from the papillary region of the human kidney and methods of isolating the same. Also described are methods for culturing, characterizing, and differentiating the same, including methods for identifying human renal stem cells that are positive for Nestin and CD133, and methods for allowing the cells to differentiate into neurons.
Chang, et al., (1987), Cancer Res., 47:1634-1645 teach a method of fetal renal stem cell isolation, based on the cell's contact insensitivity.
Gibson-D'ambrosio et al [In Vitro Cell Dev Biol. 1987 April; 23(4):279-87]teach heterogenic population of cells which may comprise renal stem cells. It is stated that these cells in culture are proximal tubule epithelial cells, indicating that these are in fact differentiated cells and not stem cells.
WO/2005/021738 teaches methods for isolation of kidney stem cells, cells isolated by the methods, and therapeutic uses for those cells. More specifically, the invention relates to isolated kidney-derived progenitor cells that have the potential to differentiate to form cells of any one or all three germ cell layers (endoderm, mesoderm, ectoderm), as well as methods for isolating the cells and for inducing specific differentiation of the cells isolated by the method, and specific markers that are present in these cells such as proteins and transcription factors. Also described are NCAM negative cells.